The formation of a thrombus in the coronary vessel is the triggering event of an unstable coronary heart disease. In patients with an unstable coronary heart disease, the central role of the activation of platelets is further enhanced by thromboxane and prostaglandin metabolites that are released from the platelets. Thus, the activation of platelets is a general therapeutic goal. Until now, such therapies comprised the use of aspirin, tienopyridines and a direct glycoprotein IIb/IIIa-inhibitor. Nevertheless, until today, a reliable biochemical marker for the activation of platelets could not be identified. Results with P-selectin, the yet most promising marker for an activation of platelets, until today, are controversial.
Individuals that suffer from a cardiovascular disease can be grouped into individuals that do not exhibit symptoms, and those that exhibit chest pain. The latter group can be grouped into individuals that exhibit a stable angina pectoris (SAP), and those with acute coronary syndromes (ACS). ACS patients can exhibit an unstable angina pectoris (UAP), or these patients already suffered from a myocardial infarction (MI). The MI can be an ST-elevated MI or a non ST-elevated MI. The occurrence of an MI can be associated by a left ventricular dysfunction (LVD). Finally, LVD patients experience a congestive heart failure (CHF) with a mortality rate of about 15%, or do not exhibit any symptoms.
Patients that are admitted with chest pain are analysed for an ST increase or depression. If this is the case, the individual will remain in hospital with a probability of nearly 100%. Since not all individuals with an MI exhibit ST-abnormalities, the troponin (TnT) level is determined, which in case of extraordinary high values indicates a high probability that an MI has occurred.
Recent progresses in the basic research have established a fundamental role for the inflammation in the mediation of all phases of the arteriosclerosis, from its beginning through progression, and, finally, to the thrombotic complications of arteriosclerotic lesions [Libby P, Ridker P M, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105(9): 1135-43. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999; 340(2):115-26. Davies M J, Thomas A C. Plaque fissuring—the cause of acute myocardial infarction, sudden ischemic death, and crescendo angina. Br Heart J 1985; 53(4):363-73. Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995; 91(11):2844-50.]. The results obtained from the association between inflammation and arteriosclerosis form the rational for the use of circulating inflammatory markers as potential predictive instruments in patients with acute coronary syndromes. Indeed, elevated levels of inflammatory markers, such as, for example, high sensitive C-reactive protein (hsCRP), serum amyloid A, and interleukin-6 (IL-6) are not only associated with acute coronary syndromes in general [Berk B C, Weintraub W S, Alexander R W. Elevation of C-reactive protein in “active” coronary artery disease. Am J Cordial 1990; 65(3):168-72., Biasucci L M, Vitelli A, Liuzzo G, et al. Elevated levels of interleukin-6 in unstable angina. Circulation 1996; 94(5):874-7.], but—what is more important—can also predict a statement regarding the clinical outcome of patients with acute coronary syndromes [Liuzzo G, Biasucci L M, Gallimore J R, et al. The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina. N Engl J Med 1994; 331(7):417-24., Biasucci L M, Liuzzo G, Grillo R L, et al. Elevated levels of C-reactive protein at discharge in patients with unstable angina predict recurrent instability. Circulation 1999; 99(7):855-60., Toss H, Lindahl B, Siegbahn A, Wallentin L. Prognostic influence of increased fibrinogen and C-reactive protein levels in unstable coronary artery disease. FRISC Study Group. Fragmin during Instability in Coronary Artery Disease. Circulation 1997; 96(12):4204-10.]. Although the “classical” acute phase protein hsCRP is regarded as the most promising biomarker for clinical uses, a substantial heterogeneity exists regarding the prevalence of elevated hsCRP levels in patients with acute coronary syndromes [Biasucci L M, Liuzzo G, Colizzi C, Rizzello V. Clinical use of C-reactive protein for the prognostic stratification of patients with ischemic heart disease. Ital Heart J 2001; 2 (3): 164-71.].
Thus, more than 30% of the patients with severe unstable angina do not exhibit elevated hsCRP levels [Liuzzo G, Biasucci L M, Gallimore J R, et al. The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina. N Engl J Med 1994; 331(7): 417-24., Heeschen C, Hamm C W, Bruemmer J, Simeons M L. Predictive value of C-reactive protein and troponin T in patients with unstable angina: a comparative analysis. CAPTURE Investigators. Chimeric c7E3 Anti-Platelet Therapy in unstable angina refractory to standard treatment trial. J Am Coll Cardiol 2000; 35(6): 1535-42.]. In addition, individual differences in the extent of the response to certain inflammatory stimuli possibly can affect the levels of the “downstream” acute-phase reactants, such as hsCRP [Pepys M B, Hirschfield G M. C-reactive protein and its role in the pathogenesis of myocardial infarction. Ital Heart J 2001, 2(11): 804-6., Liuzzo G, Biasucci L M, Rebuzzi A G, et al. plasma protein acute-phase response in unstable angina is not induced by ischemic injury. Circulation 1996; 94(10): 2373-80.]. Therefore, still an important challenge exists to identify proximal stimuli for vascular inflammation that can be used as risk-predicting serum markers in patients with coronary arteriosclerosis.
In the meantime, there is increasing evidence that the CD40-CD40L-system plays an important role in the pathophysiology of patients with unstable coronary heart disease. Apart from the cell-associated form, CD40L also occurs in a soluble biologically completely active form, namely sCD40L. sCD40L is shedded by stimulated lymphocytes and actively released upon platelet stimulation. sCD40L acts pro-inflammatory on endothelial cells and promotes the coagulation, in that monocytes and endothelial cells are stimulated to express tissue factors. In addition, sCD40L contains a KGD-sequence, a known binding motif that is specific for the predominant platelet-integrin αIIβ3. CD40L indeed is a αIIβ3-ligand, a platelet agonist, and is required for the stability of arterial thrombi. An increase of sCD40L can be detected in serum of patients with acute coronary syndromes. It was reported (Schonbeck U, Varo N, Libby P, Buring J, Ridker P M. Circulation 2001; 104: 2266-8) that apparently healthy women which exhibited elevated plasma concentrations of sCD40L, at the same time, carried an increased risk for cardiovascular events.
New findings show that a rupture of plaques and the subsequent formation of a thrombus in patients with acute coronary syndromes can lead to an activation of the exposure of CD40L in circulating platelets (Lee Y, Lee W H, Lee S C, Ahn K J, Choi Y H, Park S W, Seo J D, Park J E. Cardiology. 1999; 92: 11-6). In addition, increased concentrations of sCD40L were detected in patients with angina, whereby the concentrations were particularly high in patients with unstable angina (Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, Solum N O, Forfang K, Froland S S, Gullestad L. Circulation 1999; 10: 614-20). These results suggest that a CD40L-CD40-interaction plays an important role during the pathogenesis of arteriosclerotic processes and the development of coronary syndromes.
Establishing the correct diagnosis associated with a suitable treatment of patients with acute coronary syndromes that are not associated with an elevation of the ST-stretch can be very cumbersome. The exclusion of acute myocardial infarction in accordance with actual standards is unsatisfactory. In the last years, a focussing on the risk stratification and control of the treatment has occurred with the aim to identify patients wherein the risk exists to develop a life-threatening cardiologic event, and which, in particular, benefit from improved therapeutic and intervening strategies (Hamm C W, Bertrand M, Braunwald E. Lancet 2001; 358:1533-8). In this respect, the ECG has only limited prognostic relevance since important abnormities are rare and their detection is sparsely sensitive and specific (Kaul P, Fu Y, Chang W C, et al., J Am Coll Cardiol 2001; 38:64-71, and Savonitto S, Ardissino D, Granger C B, et al. JAMA 1999; 281: 707-13). Thus, markers of a necrosis of myocardial cells, in particular cardiac troponines, have developed into valuable tools in the evaluation of patients with acute coronary syndromes (Hamm C W, Braunwald E. Circulation 2000; 102: 118-22). Nevertheless, troponines are not actively involved in the pathophysiology of acute coronary syndromes, but rather represent a kind of surrogate markers for the fragile thrombus formation (Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L. J Am Coll Cardio. 2001; 38: 979-86, Heeschen C, van Den Brand M J, Hamm C W, Simoons M L. Circulation 1999; 100: 1509-14; Benamer H, Steg P G, Benessiano J, et al. Am Heart J 1999; 137: 815-20). In this respect, the ECG has only limited prognostic relevance since important abnormities are rare and their detection is sparsely sensitive and specific (Kaul P, Fu Y, Chang W C, et al., J Am Coll Cardiol 2001; 38:64-71 and Savonitto S, Ardissino D, Granger C B, et al. JAMA 1999; 281:707-13).
Markers of an activation of platelets that determine the activity of the disease, preferably before a myocardial necrosis occurs, could represent important additional information for the diagnostic and therapeutic stratification in patients with acute coronary syndromes. There is increasing evidence that also the CD40-ligand (CD40L, recently renamed into CD154) plays an important role in the development of the disease and plaque-destabilisation (Mach F, Schonbeck U, Sukhova G K, Atkinson E, Libby P. Nature 1998; 394: 200-3 and Lutgens E, Gorelik L, Daemen M J, et al. Nat Med 1999; 5: 1313-6). The CD40-CD40L-system is common in a multitude of leukocytes and non-leukocytic cells, including endothelial cells and smooth muscle cells (Schonbeck U, Libby P. Cell Mol Life Sci 2001; 58: 4-43), as well as in activated platelets (Henn V, Slupsky J R, Grafe M, et al. Nature 1998; 391: 591-4). In addition to the cell-associated 39-kDa form, CD40L also occurs in a soluble biologically completely active form, namely sCD40L (Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek R A. Eur J Immunol 1995; 25: 1749-54). sCD40L is shedded by stimulated lymphocytes and is actively released upon activation of platelets (Lee Y, Lee W H, Lee S C, et al. Cardiology 1999; 92: 11-6, and Henn V, Steinbach S, Buchner K, Presek P, Kroczek R A. Blood 2001; 98: 1047-54). sCD40L acts pro-inflammatory on endothelial cells and promotes the coagulation by inducing the expression of tissue factors by monocytes (Mach F, Schonbeck U, Bennefoy J Y, Pober J S, Libby P. Circulation 1997; 96: 396-9) and endothelial cells (Urbich C, Mallat Z, Tedgui A, Clauss M, Zeiher A M, Dimmeler S. J Clin Invest 2001; 108: 1451-8). In addition, sCD40L contains a KGD-sequence (Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek R A. Eur J Immunol 1995; 25: 1749-54), a known binding motif that is specific for the predominant platelet-integrin αIIbβ3 (Scarborough R M, Naughton M A, Teng W, et al. J Biol Chem 1993; 268: 1066-73). It could be shown that CD40L indeed represents an αIIbβ3-ligand, a platelet agonist, and is required for the stability of arterial thrombi (Andre P, Prasad K S, Denis C V, et al. Nat Med 2002; 8: 247-52).
These findings stringently show that sCD40L plays an important role in the pathophysiology of acute coronary syndromes. Interestingly, an increase of sCD40L can be detected in serum of patients with acute coronary syndromes (Aukrust P, Muller F, Ueland T, et al. Circulation 1999; 100:614-20). It was reported that apparently healthy women that exhibited elevated plasma concentrations of sCD40L carried an increased risk for cardiovascular events (Schonbeck U, Varo N, Libby P, Buring J, Ridker P M. Circulation 2001; 104: 2266-8). It is the aim of the present invention to examine the predictive value of sCD40L-concentrations with respect to cardiac events and the curing effects of the glycoprotein IIb/IIIa-inhibitor abciximab in patients with acute coronary syndromes, wherein the data base of the CAPTURE-study (c7E3 Anti-Platelet Therapy in Unstable Refractory angina) was used (CAPTURE. Lancet 1997; 349:1429-35).
Inflammatory markers that determine the activity of the disease, possibly before a myocardial necrosis occurs, can represent important additional information for the diagnostic and therapeutic stratification in patients with acute coronary syndromes. The specific therapeutic inhibition of cytokines that are essential for the plaque-stability may be a novel strategy for the treatment of patients with unstable and stable coronary heart disease.
It was recently shown for placental-growth factor (PlGF), a member of the family of the vascular-endothelial-growth factor-family (VEGF-family) of growth factors, that it is up-regulated in early and progressed arteriosclerotic lesions.
U.S. Pat. No. 6,225,088 describes PlGF in connection with proliferative diseases, whereas in the WO 92/06194, PlGF is described as an angiogenetic factor.
De Falco et al. (De Falco S, Gigante B, Persico M G. “Structure and function of placental growth factor” Trends Cardiovasc Med August 2002; 12(6):241-6) describe the association of derogated angiogenesis and arteriogenesis during pathologic conditions, such as, for example, ischemia and tumour formation in mice. Thereby, PlGF is described as an essential factor for the angiogenesis under pathologic conditions. PlGF is proposed as an alternative target for an angiogenetic therapy.
Luttun et al. (Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy J A, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert J M, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak H F, Hicklin D J, Carmeliet P. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumour angiogenesis, arthritis and atherosclerosis by anti-Flt1 Nat Med August 2002; 8(8): 831-40) describe therapeutic methods of PlGF and Flt-1 in the context of angiogenesis. It is furthermore described that inhibition of PlGF reduces the growth and vulnerability of arteriosclerotic plaques.
Oura et al. (Oura H, Bertoncini J, Velasco P, Brown L F, Carmeliet P, Detmar M. A critical role of placental growth factor in the induction of inflammation and oedema formation. Blood Jan. 15, 2003; 101(2): 560-7) describe the role of PlGF in cutaneous inflammation and angiogenesis. Furthermore, the effects of elevated and reduced levels of PlGF and their comparison with the status of the inflammation are described.
The pro-inflammatory cytokine CD40L is released by activated platelets. The soluble form of CD40L, namely sCD40L, is increasingly present in patients with acute coronary syndromes. Thus, the prognostic value of sCD40L as a marker for the activation of platelets was also examined in view of the therapeutic effect of an inhibition of the glycoprotein IIb/IIIa-receptor.
Pregnancy-associated plasma protein-A (PAPP-A) is a high molecular weight zinc-binding matrix-metalloproteinase, belonging to the metzincin-superfamily of metalloproteinases, and was initially identified in the plasma of pregnant women. It is broadly used for the screening of foetal trisomy in the first trimester of gestation. It was also recently found for PAPP-A that it is expressed in eroded and loosened plaques, respectively, but is only minimally expressed in stable plaques, and in those wherein a re-occurrence with symptoms as in patients with ACS is assumed. Nevertheless, the exact role of circulating PAPP-A plasma levels for the prediction of hard endpoints, such as death or myocardial infarction, is not exactly determined in patients with ACS. In addition, it is completely unknown whether PAPP-A plasma levels provide additional prognostic information in patients with ACS compared to recently established biomarkers. Therefore, the inventors compared the prognostic significance of PAPP-A plasma levels with markers of systemic inflammation, the activation of platelets, ischemia, and myocardial necrosis in patients with ACS.
Many different molecular markers can be found in the state of the art that can be suitable for the diagnosis of a cardiovascular disease. Examples of such markers are, amongst others:
Pregnancy-associated plasma protein A (PAPP-A); C-reactive protein (CRP); hs-CRP; placental growth factor (PlGF); interleukin-18 (IL-18IL-18b); brain natriuretic peptide (BNP); NT-pro brain natriuretic peptide (NT-proNP); sCD40L, cTnI/T, IL-10, ICAM-1, VCAN-1, E-selectin, P-selectin, IL-6, VEGF, serum amyloid A (SAA), CKMB, MPO, LpPLAz, GP-BB, IL1RA, TAF1, soluble fibrin, anti-oxLDL, MCP-1, tissue factor (TF), MMP-9, Ang-2, Tffi-2, IL-P, bFGF, PCM, and VEGF-A.
Some of the above indicated markers are markers known and characterised for the examination of coronary diseases, nevertheless, others have not yet been correspondingly examined. Nearly all of the given markers have a diagnostic value with respect to certain cardiovascular events. TnT, for example, is of particular value for the diagnosis and the prediction of MI (see above). Inflammatory markers, such as CRP, are valuable for the diagnosis and prediction of an inflammation that can lead to a plaque-rupture and MI.
Many of the above indicated markers have a diagnostic value for cardiovascular diseases. The use of a combination of markers was described only very conservatively.
Lund et al. in Circulation. 2003, 108:1924-1926, describe the combination of PAPP-A with TnI (troponin I); Peng et al. in Clinica Chimica Acta 319 (2002) 19-26, describe the combination of sCD40L with sICAM-1 and sVCAM-1. Lenderink et al. in European Heart Journal (2003) 24, 77-85, describe the combination of TnT with CRP. Heeschen et al. in Journal of the American College of Cardiology; Vol. 35, No. 6, 2000, mention the combination of TnT with CRP.
Heeschen et al. in Circulation. 2003; 107:2109-2114, describe the combination of TnT, CRP and IL-10. Blankenberg et al. in Circulation. 2002; 106:24-30, describe the combination of CRP and IL-6. Autiero et al. in Journal of Thrombosis and Haemostasis, 1:1356-1370, describe the combination of PlGF and VEGF.
However, until now the potential of the improved analysis by the combination has neither been examined, nor was the development of selective superior marker-assays completed. Which markers are suitable for an effective diagnosis therefore can not be readily derived from the above indicated publications.
In view of the above, it is therefore an object of the present invention to provide a method by which the risk of an adverse cardiovascular event being due to coronary thrombosis can be estimated with the aid of an individual risk profile. It is a further object of the present invention to develop a method for the evaluation of the probability, whether a treatment with an active ingredient for the inhibition of the placental growth factor (PlGF) is advantageous. With the aid of this method the attending physician shall be, better than before, enabled to select suitable measures in order to positively influence the patients and/or to prevent an adverse event, or to at least reduce it in its severity for the affected patients.
It is the object of the present invention to develop a method by which the risk to suffer from an adverse cardiovascular event being due to a coronary thrombosis can be estimated with the aid of an individual risk profile. This shall be done by measuring the concentration of a marker of the activation of platelets. It is furthermore an object of the present invention to develop a method for evaluating the probability, whether a treatment with an active ingredient for the inhibition of the activation of platelets is advantageous. With the aid of this method the attending physician shall be, better than before, enabled to select suitable measures in order to positively influence the patients and/or to prevent an adverse event, or to at least reduce it in its severity for the affected patients.
It is a further object of the present invention to find general combinations of diagnostic markers for cardiovascular events that allow for a precise diagnosis which cardiovascular event the patient had already suffered from, and from which he will possibly suffer in the future. It is a particular object to find marker combinations that can advantageously be performed in parallel. Ideally, these measurements would allow for measuring patients with chest pain, simultaneously with TnT.
The object of the present invention is solved by a method for analysing samples in assiciation with acute cardiovascular diseases. The method according to the invention comprises the steps of: (a) obtaining a biological sample to be analysed from a subject; (b) determining of the concentration of at least one marker selected from soluble CD40-ligand (sCD40L), PAPP-A, and PlGF, (c) optionally, determining of the concentration of at least one additional marker selected from troponin T (TnT), MPO, NT-proBNP, VEGF, BNP, and additional inflammatory markers, and (d) comparing the results that are obtained for the sample to be analysed with reference value/s and/or the values from reference samples.
The markers that are used for the analysis in the context of the present invention are, to the largest part, markers that are well known and characterised from the state of the art, that, however, until now were only insufficiently characterised for the use in a diagnosis of an acute cardiovascular disease.
Thus, C-reactive protein (CRP) and hsCRP are characterised as markers of systemic inflammation, troponin cTnl/T is characterised as marker for necrosis; the pregnancy associated plasma protein A (PAPP-A) is characterised as a marker for the activation of macrophages; IL-10 (Interleukin 10) is characterised as a marker for the inflammatory balance, sCD40L is characterised as a marker for the thrombo-inflammatory activation, MPO (myeloperoxidase) is characterised as a marker for oxidative stress, placental growth factor (PlGF) is characterised as a marker for vascular inflammation, and the markers brain natriuretic peptide (BNP) and NT-pro brain natriuretic peptide (NT-proNP) are characterised as markers of neurohumoral activation and ischemia.
Other similar and also useful markers are interleukin-18 (IL-18/IL-18b), ICAM-1, VCAN-1, E-selectin, P-selectin, IL-6, VEGF, serum amyloid A (SAA), CKMB, LpPLAz, GP-BB, IL-1RA, TAF-1, soluble fibrin, anti-oxLDL, MCP-1, tissue factor (TF), MMP-9, Ang-2, Tffi-2, bFGF, PCM, and VEGF-A.
WO 03/040692 (and Circulation 2001; 104:2266-2268) describes that sCD40L can be advantageously used in combination with an inflammatory marker, in particular CRP. However, this combination is exclusively used for the diagnosis of non-acute cardiac diseases, due to doubtful results a stratification is rejected by WO 03/040692.
Preferred is a method according to the invention, wherein the sample to be analysed and/or the reference sample is derived from a mammal, in particular from a human. Further preferred is a method according to the invention, wherein the sample to be analysed and/or the reference sample is selected from the group consisting of peripheral blood or fractions thereof, and cell culture suspensions or fractions thereof. It if further preferred that the sample to be analysed and/or the reference sample is blood serum or blood plasma. Peripheral whole blood is particularly preferred as a sample to be analysed and/or reference sample.
According to a further aspect of the method according to the invention, the sample to be analysed and/or the reference sample can be pre-treated, wherein, e.g., a coagulation inhibitor, in particular heparin, is added to said peripheral blood.
In accordance with the invention, it could surprisingly be found that, in addition to the individual analysis of several of the above-mentioned markers, also combinations of such markers are possible for a diagnosis or monitoring of acute cardiac events, allowing for a markedly improved analysis. In doing so, combinations of markers could be identified as particularly preferred that relate to different aspects of the adverse cardiac event, but can be analysed simultaneously (that is, simultaneously or in a more or less timely spaced series of measurements). In one aspect of this invention, additional markers are selected from inflammatory markers, such as, for example, from CRP, (hs)CRP, and IL-10.
According to a further aspect of the method according to the invention, the markers and combinations thereof as analysed are selected from sCD40L; PAPP-A; PlGF; sCD40L+TnT; PAPP-A+TnT; PlGF+TnT; sCD40L+PAPP-A; sCD40L+PlGF; PAPP-A+PlGF; sCD40L+PAPP-A+TnT; sCD40L+PlGF+TnT; PAPP-A+PlGF+TnT; sCD40L+PAPP-A+PlGF; and sCD40L+PAPP-A+PlGF+TnT. Then, preferred is the further combination with at least one of the additional markers MPO, NT-proBNP, BNP, CRP, (hs)CRP, and IL-10.
Then, particularly preferred according to the invention is a method, wherein the markers and combinations thereof as analysed are selected from CRP, TnT, PAPP-A; CRP, TnT, PAPP-A, IL-10; CRP, TnT, PAPP-A, IL-10, sCD40L, and TnT, PAPP-A, IL-10, sCD40L, VEGF.
The method as used according to the invention for determining the concentrations of the markers as analysed can be selected from all suitable methods for detecting proteins in biological samples that are known to the person of skill in the art. In the context of the present invention, the actual method is not important as long as the method is sensitive enough in order to fall below the detection level that is required for an accurate determination of the concentrations of the markers. Suitable methods are normally based on the binding of a label to the marker to be determined, and the subsequent detection of this label. Thereby, the binding can be covalent or non-covalent and/or occur directly or indirectly. Suitable methods for measuring according to the present invention include, e.g., electro-chemiluminescence. Turbidimetry, nephelometry, and latex-enhanced turbidimetry or nephelometry can also be used.
Due to its high sensitivity and the fact that these methods can also be adapted to high-throughput-environments, according to the invention methods are preferred, wherein the determining of the concentration takes place by means of an immunological method by means of marker-binding molecules. Examples for such methods are ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassays or solid-phase immunoassays. Preferred is therefore that the marker-binding molecules are selected from the group consisting of anti-marker-antibodies or parts thereof, and marker-receptors or parts thereof.
These molecules can be selected from a very large multitude of marker-specific molecules. It is preferred that the marker-binding molecules are selected from the group consisting of antibodies that are specifically directed against markers or against parts thereof, or parts or fragments thereof, and a marker-receptor or parts thereof, or an integrin, e.g. the platelet-integrin (αIIbβ3 or parts thereof. Particularly preferred is a method according to the invention, wherein the antibodies, parts or fragments thereof comprise polyclonal antibodies, monoclonal antibodies, Fab-fragments, scFv-antibodies, and diabodies.
According to a further aspect of the method of the present invention components of the method can be present bound to a solid phase, thus, the marker-binding molecules can be present in solution or matrix-immobilised. A multitude of materials that are known to the person of skill are used as matrices, such as, for example, resin-matrices and/or common column-matrices. Particularly preferred is furthermore a method according to the invention, wherein the marker-binding molecules are coupled to one or several detection molecules from the group consisting of fluorescein thioisocyanate, phycoerythrine, enzymes (for example horse-radish-peroxidase), and magnetic bead.
According to a further aspect of the method according to the invention, the marker-binding molecules can be detected with an antibody that is coupled to one or several detection molecules. Thus, this represents an indirect detection of the binding of the molecule. Such two-step detections are very well known to the person of skill, for example, from the technology of anti-antibody-detection.
According to a further aspect of the method of the present invention, immunocytological methods can be used for the analysis of the sample. For this, all methods are suitable that allow for a specific determination based on the marker/molecule-interaction. Preferred are methods that are selected from the group consisting of sandwich-enzyme-immunoassay, ELISA, and solid phase immunoassays.
The results that are obtained for the samples to be analysed are usually compared with a reference sample. Which sample can serve as a reference sample will, in particular, depend from the kind of the sample that is analysed, and the history of the disease of the individual from which the sample to be analysed is derived. Preferred is a method according to the invention, wherein the reference sample is derived from one or the mean value of several mammals, wherein a cardiovascular disease was excluded. Nevertheless, this is not mandatory, if, e.g. the progression of a disease shall be determined, also an “old” sample of the same patient can be used as a reference sample. It will be obvious for the person of skill, which samples are suitable as reference samples for the method according to the invention.
According to a further aspect of the method according to the present invention, the acute cardiovascular diseases that are to be diagnosed and/or prognosed, and/or whose therapy shall be monitored can be selected from the group consisting of unstable angina, myocardial infarction, acute heart syndromes, coronary arterial disease, and heart insufficiency. Nevertheless, it shall not be excluded that the method according to the invention is suitable for and can be employed in additional acute cardiac disease conditions.
A further aspect of the present invention relates to a diagnostic kit, wherein said kit comprises means for performing the method according to the invention, optionally together with additional components and/or excipients. Preferably, such means are at least one antibody for detecting of markers, and means for the subsequent quantification of said markers. In addition, the kit can contain other components and/or enzymes for performing the method according to the present invention, e.g. instruction manuals for an interpretation of the results of the assays in view of the risk profile of the patient, and corresponding countermeasures and proposals for therapy.
It is preferred to perform the method according to the invention with the aid of a diagnostic kit that comprises gold-labelled polyclonal mouse-indicator antibodies, biotinylated polyclonal detection antibodies, and an assay device comprising a fibreglass-fleece.
A further aspect of the present invention thus relates to the use of the method according to the invention for the diagnosis and/or prognosis of acute cardiovascular diseases, and/or for monitoring of their therapy. This is done by the quantitative and critical determination of markers. Based on the risk profile that then can be generated, suitable countermeasures can then be performed by the attending physician in order to positively influence the patients and to prevent the adverse event or at least to reduce it in its severity for the affected patient. Such a therapy according to the invention can, e.g., comprise the administration of statins or inhibitors of the glycoprotein IIb/III-receptor, in particular abciximab. However, the person of skill is aware of further possible therapies in accordance with the common scheme in order to treat cardiovascular diseases that can occur.
In a further embodiment of the invention, an anti-inflammatory means is co-administered. Said means can be selected from non-steroid or steroid anti-inflammatory means that, e.g., can include: alclofenac; alclometason; dipropionate; algestonacetonide; alpha-amylase; amcinafal; amcinafid; amfenac sodium; amiprilose hydrochloride; anakinra; anirolac; anitrazafen; apazon; balsalazid disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelain; broperamol; budesonide; carprofen; cicloprofen; cintazon; cliprofen; clobetasolpropionate; clobetasonbutyrate; clopirac; cloticasonpropionate; cormethasonacetate; cortodoxon; deflazacort; desonid; desoximetason; dexamethasondipropionate; diclofenac potassium; diclofenac sodium; diflorasondiacetate; diflunudon sodium; diflunisal; difluprednat; diftalon; dimethylsulfoxide; drocinonid; endryson; enlimomab; enolicam sodium; epirizol; etodolac; etofenamat; felbinac; fenamol; fenbufen; fenclofenac; fenclorac; fendosal; fenpipalon; fentiazac; flazalon; fluazacort; flufenamine acid; flumizol; runisolidacetate; plunixin; flunixin meglumine; fluocortin butyl; fluorometholonacetate; fluquazon; flurbiprofen; fluretofen; fluticasonpropionate; puraprofen; furobufen; halcinonid; halobetasolpropionate; halopredonacetate; ibufenac; ibuprofen; ibuprofen aluminium; ibuprofen piconol; ilonidap; indomethacin; indomethacin sodium; indoprofen; indoxol; mitrazol; isoflupredonacetate; isoxepac; isoxicam; ketoprofen; lofemizol hydrochloride; lomoxicam; loteprednol etabonat; meclofenamat sodium; meclofenamine acid; meclorison dibutyrate; mefenamin acid; mesalamine; meseclazon; methylprednisolon suleptanate; momiflumat; nabumeton; naproxen; naproxen sodium; naproxol; nimazon; olsalazin sodium; orgotein; orpanoxin; oxaprozin; oxyphenbutazon; paranylin hydrochloride; pentosan polysulfat sodium; phenbutazon sodium glycerate; pirfenidon; piroxicam; piroxicam cinnamate; piroxicam olamine; pirprofen; prednazat; prifelon; prodolinic acid; proquazon; proxazol; proxazolcitrate; rimexolon; romazarit; salcolex; salnacedin; salsalat; salicylates; sanguinariumchloride; seclazon; sermetacin; sudoxicam; sulindac; suprofen; talmetacin; talniflumat; talosalat; tebufelon; tenidap; tenidap sodium; tenoxicam; tesicam; tesimid; tetrydamine; tiopinac; tixocortol pivalat; tolmetin; tolmetin sodium; triclonid; triflumidate; zidometacin; glucocorticoides; zomepirac sodium.
In the context of the present invention, “diagnosis” relates to the ascertaining, whether an individual has suffered from a particular cardiovascular event. In the context of the present invention, “prognosis” relates to the prediction of the probability (in %) whether an individual will suffer from a particular cardiovascular event. In the context of the present invention, “stratification of the therapy” relates to the determination of the suitable therapeutic treatment for said cardiovascular event that will occur or has occurred. In the context of the present invention, “monitoring of the therapy” relates to the control and, optionally, an adjusting of the therapeutic treatment for an individual. In the context of the present invention, “therapeutic treatment” includes all treatments that possibly improve the pathophysiological condition of an individual, and, e.g., includes the administration of pharmaceutics as well as chirurgical treatment (e.g. balloon dilatation).
Base line-values of the sCD40L-concentration make available information of prognostic value in patients with acute coronary syndromes, independently of the occurrence of a myocardial necrosis. In addition, the high risk group of patients that can gain the largest benefit from an anti-platelet-treatment with abciximab can be identified by means of sCD40L.
The present study provides direct evidence for the fact that sCD40L is an informative biochemical marker of an activation of platelets. An increase of the concentration of CD40L reliably identifies a specific subgroup of patients with acute coronary syndromes that carry a profound risk to suffer from a cardiac event, and that gain the largest benefit from treatment with the glycoprotein IIb/IIIa-receptor-antagonist abciximab. Correspondingly, CD40L does not only essentially contribute to the pathophysiology of acute coronary syndromes, but provides a reliable and informative clinical marker by which patients having a formation of high risk-lesions and/or coronary thrombosis can be identified (Andre P, Prasad K S, Denis C V, et al., Nat Med 2002; 8:247-52; Andre P., Nannizzi-Alaimo L, Prasad S K, Phillips D R.; Circulation 2002; 106: 896-9).
In 40.5% of the CAPTURE-patients, the sCD40L-concentrations in the circulation were present above the calculated threshold concentration of 5.0 μg/l. These patients having an elevated sCD40L-concentrations carry a profound cardiac risk to experience a lethal or non-lethal myocardial infarction. This elevated cardiac risk in patients with high sCD40L-concentrations receiving a placebo was particularly obvious during the first 72 hours (FIG. 5a). However, the frequency of the events during the whole six months developed further apart (FIG. 5b). sCD40L could be found as an informative prognostic marker being independent from the detection of a myocardial necrosis and independent from the inflammatory markers CRP and TNF-α as well as from the adhesion molecule ICAM-1. TnT, CRP, and sCD40L provided reliable prognostic information in a multivariate regression model (Table 2). Using the sCD40L-concentration, patients without indications of a myocardial necrosis were identified that exhibited an increased cardiac risk. By using predetermined threshold concentrations for TnT and CRP together with a classification of the patients based on the number of cardiac markers that were elevated it was found that a simultaneous estimation of these pathobiologically different biochemical markers at the time where the patient is admitted allows for an informative prediction of the risk of the patient to suffer from an adverse cardiac event during the following six months.
Troponines represent markers of a myocardial necrosis, nevertheless, they are not actively involved in the pathophysiology of acute coronary syndromes. Rather, they are surrogate markers for the fragile formation of thrombi (Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L; J Am Coll Cardiol 2001; 38:979-86; Heeschen C, van Den Brand M J, Hamm C W, Simoons M L; Circulation 1999; 100:1509-14; Benamer H, Steg P G, Benessiano J, et al.; Am Heart J 1999; 137:815-20). In post mortem-studies on patients with acute coronary syndromes, an erosion or rupture of the fibrous caps of the arteriosclerotic plaques that are the basis of the pathophysiology was identified (Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L; J Am Coll Cardiol 2001; 38:979-86; Heeschen C, van Den Brand M J, Hamm C W, Simoons M L; Circulation 1999; 100:1509-14). An exposure of components of the plaques, collagen and other components of the vascular wall leads to an increase of the vascular tonus and the activation of platelets (Farb A, Burke A P, Tang A L, et al.; Circulation 1996; 93:1354-63; Davies M J, Thomas A C; Br Heart J 1985; 53:363-73; Davies M J; N. Engl J Med 1997; 336:1312-4). The thrombotic embolism of a coronary artery with increased microvascular perfusion and necrosis is an essential component of acute coronary syndromes (Heeschen C, van Den Brand M J, Hamm C W, Simoons M L; Circulation 1999; 100:1509-14; Benamer H, Steg P G, Benessiano J, et al.; Am Heart J 1999; 137:815-20). Correspondingly, sensitive markers, in particular troponines as surrogate markers of an arterial thrombotic embolism that results from an active thrombotic process in the underlying lesion, serve for the detection of a small injury of the myocardium.
In contrast to this, sCD40L could be directly involved in the pathophysiology of acute coronary syndromes in several ways. Recent evidences suggest that CD40L contributes essentially to the progression of an arteriosclerosis, and correspondingly to a destabilisation of arteriosclerotic plaques (Mach F, Schonbeck U, Sukhova G K, Atkinson E, Libby P; Nature 1998; 394:200-3; Lutgens E, Gorelik L, Daemen M J, et al.; Nat Med 1999; 5:1313-6). It was proposed that CD40/CD40L-interactions promote complications by atheroms in that they induce the expression of cytokines, chemokines, growth factors, matrix-metalloproteinases, and pro-coagulants in different atherom-associated cellular types (Schonbeck U, Libby P.; Cell Mol Life Sci 2001; 58:4-43; Henn V, Slupsky J R, Grafe M, et al.; Nature 1998; 391:591-4; Henn V, Steinbach S, Buchner K, Presek P, Kroczek R A; Blood 2001; 98:1047-54; Mach F, Schonbeck U, Bonnefoy J Y, Pober J S, Libby P.; Circulation 1997; 96:396-9; Miller D L; Yaron R, Yellin M J; J Leukoc Biol 1998; 63-373-9; Kotowicz K, Dixon G L, Klein N J, Peters M J, Callard R E; Immunology 2000; 100:441-8). Novel studies have shown that, in addition to leukocytes and non-leukocytic cells including granulocytes, mononuclear phagocytes, endothelial cells and cells of the smooth musculature (Schonbeck U, Libby P; Cell Mol Life Sci 2001; 58:4-43), activated platelets produce and release large amounts of sCD40L (Henn V, Steinbach S, Buchner K, Presek P, Kroczek R A; Blood 2001; 98:1047-54). Another study shows that a cardiopulmonary bypass causes an increase of the concentration of sCD40L in plasma that corresponds to a decrease of the content of CD40L in platelets, suggesting that sCD40L primarily is derived from the platelets and could contribute to the thrombotic complications that are related to such a bypass (Nannizzi-Alaimo L, Rubenstein M H, Alves V L, Leong G Y, Phillips D R, Gold H K; Circulation 2002, 105:2849-2854). It was furthermore found that sCD40L positively correlates with soluble P-selectin in plasma and 11-dehydro-thromboxane B2-concentrations in urine (Cipollone F, Mezzetti A, Porreca E, et al.; Circulation 2002; 106:399-402). In addition, experimental analyses showed that CD40L is required for a stabilisation of arterial thrombi (Andre P, Prasad K S, Denis C V et al.; Nat Med 2002; 8:247-52). The present study now provides direct evidence for the fact that CD40L indeed is a marker of the activation of platelets. The activation of platelets as determined by flow cytometry in patients with acute coronary syndromes correlated significantly with the concentrations of sCD40L in serum (FIG. 7). Interestingly, sCD40L was found as an independent predictive factor of an activation of platelets with the highest significance. The results of the present study established sCD40L-concentrations as highly informative prognostic markers in patients with acute coronary syndromes that are in danger to suffer from a thrombosis. These findings are supported by the fact that an inhibition of the glycoprotein IIb/IIIa-receptor by abciximab eliminated the increased risk in patients with acute coronary syndromes and elevated sCD40L-concentrations. While troponin positively indicated the tendency of a thrombus to cause an embolism and to lead to myocardial necrosis, elevated concentrations of sCD40L in patients with acute coronary syndromes reflect the thrombotic activity of the triggering lesion to recruit and activate platelets.
It was shown in an earlier analysis of a subgroup of patients of the CAPTURE-study that an additional treatment with the glycoprotein IIb/IIIa-receptor-antagonist abciximab reduced the elevated risk of troponin-positive patients to the extent of troponin-negative patients (Hamm C W, Heeschen C, Goldmann B, et al.; N Engl J Med 1999; 340:1623-9). These patients constitute about ⅓ of the patients with acute coronary syndromes (Hamm C W, Braunwald E; Circulation 2000; 102:118-22; Antman E M, Tanasijevic M J, Thompson B, et al; N Engl J Med 1996; 335:1342-9; Ohman E M, Armstrong P W, Christenson R H, et al.; N Engl J Med 1996; 335:1333-41; Hamm C W, Ravkilde J, Gerhardt W, et al.; N Engl J Med 1992; 327:146-50; Hamm C W, Goldmann B U, Heeschen C, Kreymann G, Berger J, Meinertz T; N Engl J Med 1997; 337:1648-53). Similar findings regarding troponin T and troponin I later resulted from other studies (Newby L K, Ohman E M, Christenson R H, et al.; Circulation 2001; 103:2891-6; Januzzi J L, Chae C U, Sabatine M S, Jang I K; J Thromb Thrombolysis 2001; 11:211-5; Heeschen C, Hamm C W, Goldmann B, Deu A, Langenbrink L, White H D; Lancet 1999; 354:1757-62), and troponines were subsequently included into the novel guidelines as part of the risk stratification in patients with acute coronary syndromes (Hamm C W, Bertrand M, Braundwald E; Lancet 2001; 358:1533-8; Braunwald E, Maseri A, Armstrong P W, et al.; Eur Heart J 1998; 19:D22-30). It is shown in the present study that such a pronounced positive effect of an anti-platelet-therapy is obvious also in patients with elevated sCD40L-concentrations. The present analysis suggests that patients with acute coronary syndromes that exhibit increased concentrations of sCD40L are effectively stabilised by the glycoprotein IIb/IIIa-receptor-antagonist abciximab (FIGS. 5a, b). At a calculated threshold concentration of 5.0 μg/l, an abrupt change of the risk quotient of 0.87 for the second quintile, and 1.12 for the third quintile towards significantly lower values of 0.36 for the fourth quintile, and 0.38 for the fifth quintile, respectively, (FIG. 4) was observed. Interestingly, the concentrations of TnT and sCD40L provided independent predictive values with respect to both the risk of ischemic events as well as the positive effect of a glycoprotein IIb/IIIa-receptor-inhibition by abciximab. Patients without indications of a myocardial injury (troponin-increase is lacking) which, nevertheless, exhibited increased concentrations of sCD40L, gained a substantial benefit from the treatment with the glycoprotein IIb/IIIa-inhibitor abciximab. Thus, patients with a high risk for a thrombosis of the coronary vessels, as proven either by an increase of the sCD40L-concentration or an increase of the TnT-concentration, which finally made up 54% of the overall patients involved the CAPTURE-study, had a pronounced advantage from the treatment with abciximab, with a risk quotient of 0.38[0.21-0.72]; p<0.001).
In summary, it can be stated that the present study documents the important and independent role of sCD40L as a marker of the activation of platelets for the diagnostic and therapeutic risk stratification. The increased cardiac risk of patients with high sCD40L-concentrations that received a standard therapy with heparin and aspirin was reverted by the glycoprotein IIb/IIIa-receptor-antagonist abciximab. The combined use of troponines and sCD40L that both represent essential components of the pathophysiology in patients with acute coronary syndromes provides important insights into the activity of the disease, the cardiologic risk, and the effectiveness of a treatment by means of glycoprotein IIb/IIIa-inhibition by abciximab being superior to the use of a single marker.
The results of the present study establish the PlGF serum level as a novel and effective independent prognostic determinant of the clinical outcome in patients with acute coronary syndromes. It has to be particularly noted that in patients with lower hsCRP serum levels elevated PlGF serum levels identify a subgroup of patients that suffer from a significantly increased cardiac risk (fitted hazards-ratio 3.58 [95% CI 1.48-7.72]; p=0.001).
The predictive value of PlGF serum levels is independent from evidence for myocardial necrosis as determined by the troponin serum level. Finally, elevated PlGF serum levels not only identify those patients with acute chest pain that develop acute coronary syndromes, but also those patients that suffer from an increased risk of reoccurring instability from an initial acute coronary syndrome after discharge. Thus, measuring of the PlGF serum levels can not only be a reliable and effective clinical tool for the identification of patients with high risk formation of lesions but also of persistent vascular inflammation of the coronary circulation.
The role of PlGF as a primary inflammatory marker of the instability of arteriosclerotic lesions can be explained by its well documented pro-inflammatory effects in animal models of arteriosclerosis or arthritis [Luttun A, Tjwa M, Moons L, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 2002; 8(8):831-40.]. Although PlGF belongs to the family of VEGF, its etiopathogenetic role appears to be associated rather with inflammation as with angiogenesis [Luttun A, Tjwa M, Moons L, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 2002; 8(8):831-40.]. Indeed, whilst the VEGF-increase due to hypoxia and the increase of the VEGF-serum level are regarded as an early adaptation of the myocardium to the decreasing bloodflow [Lee S H, Wolf P L, Escudero R, Deutsch R, Jamieson S W, Thistlethwaite P A. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 2000; 342(9):626-33.], PlGF is not affected or down-regulated by hypoxia [Khaliq A, Dunk C, Jiang J, et al. Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for “placental hyperoxia” in intrauterme growth restriction. Lab Invest 1999; 79(2):151-70., Cao Y, Linden P, Shima D, Browne F, Folkman J. In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. J Clin Invest 1996; 98(11):2507-11.]. In agreement with these data, the results of this study do not result in a correlation between the PlGF serum level and the troponin T serum level as a marker of myocardial necrosis, whereas the VEGF serum level positively correlated with the troponin T serum level. In agreement with this, PlGF did not correlate with the VEGF serum level.
Therefore, the PlGF serum level appears not to be affected by myocardial necrosis. In contrast, the VEGF serum levels are coupled with an increase of troponin T, affected TIMI flow, and clinical signs of myocardial ischemia [Heeschen C, Dimmeler S, Hamm C W, Boersma E, Zeiher A M, Simoons M L. Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes. Circulation 2003; 107: 524-530.]. The PlGF serum level being insensitive versus smaller myocardial injuries could specifically be important in patients with acute coronary syndromes, out of which about one third at presentation are positive for troponin [Hamm C W, Braunwald E. A classification of unstable angina revisited. Circulation 2000; 102(1):118-22.].
Similarly, a myocardial injury could also compromise the value of the hsCRP serum level in order to be able to predict the outcome in patients with acute coronary syndromes. As a classical unspecific downstream acute-phase-marker, the hsCRP serum levels in patients with myocardial injury are increased as measured by an increase of troponin T. It is well established that elevated serum levels of hsCRP are found before the occurrence of a marker of myocardial necrosis in nearly all patients in which an unstable angina occurs before an infarction [Liuzzo G, Baisucci L M, Gallimore J R, et al. Enhanced inflammatory response in patients with preinfarction unstable angina. J Am Coll Cardiol 1999; 34(6): 1696-703.]. The specificity of elevated hsCRP serum level confirming an enhanced vascular inflammation in the presence of myocardial injury is therefore very limited.
Thus, elevated hsCRP serum levels in troponin-positive patients can only represent an elevated risk secondary to myocardial injury as a surrogate marker for thrombotic embolism that rather is derived from an active thrombotic process within the culpritic lesion than from a persisting vascular inflammation.
Indeed, when each of troponin T and VEGF as markers of myocardial necrosis and ischemia are included into a multivariate analysis, elevated hsCRP serum levels are no longer predictive for an elevated risk in patients with acute coronary syndromes.
More important, the reported prevalence of elevated hsCRP serum levels varies considerably in acute coronary syndromes. (more than 30% of the patients with severe unstable angina and more than 50% of the patients with an acute myocardial infarction do not exhibit elevated hsCRP serum levels). Elevated hsCRP serum levels are lacking in more than 30% of patients with severe unstable angina and in more than 50% of those with an acute myocardial infarction that does not follow after an unstable angina [Liuzzo G, Baisucci L M, Gallimore J R, et al. Enhanced inflammatory response in patients with preinfarction unstable angina. J Am Coll Cardiol 1999; 34(6): 1696-703.] suggesting an important heterogenicity of the role of inflammatory triggers of the clinical syndrome of the coronary instability [Libby P, Ridker P M, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105(9): 1135-43.]. However, it is also well established that individuals can vary in their systemic responses towards a particular inflammatory stimulus [Liuzzo G, Buffon A, Biasucci L M, et al. Enhanced inflammatory response to coronary angioplasty in patients with severe unstable angina. Circulation 1998; 98(22):2370-6., Liuzzo G, Angiolillo D J, Buffon A, et al. Enhanced response of blood monocytes to in vitro lipopolysaccharide-challenge in patients with recurrent unstable angina. Circulation 2001; 103(18):2236-41. Biasucci L M, Vitelli A, Liuzzo G, et al. Elevated levels of interleukin-6 in unstable angina. Circulation 1996; 94(5):874-7.]. The increase in hsCRP or IL-6 that was observed in response to the vascular trauma induced by balloon dilatation or even by uncomplicated cardiac catheterisation correlates linearly with baseline hsCRP or interleukin-6 serum levels [Liuzzo G, Buffon A, Biasucci L M, et al. Enhanced inflammatory response to coronary angioplasty in patients with severe unstable angina. Circulation 1998; 98(22):2370-6.]. In addition, the IL-6 production by monocytes that are isolated from patients with unstable angina significantly increased in patients with elevated hsCRP serum levels, compared to patients with normal hsCRP serum levels [Liuzzo G, Angiolillo D J, Buffon A, et al. Enhanced response of blood monocytes to in vitro lipopolysaccharide-challenge in patients with recurrent unstable angina. Circulation 2001; 103(18):2236-41.]. These individual differences in the extent of the response to a particular inflammatory stimulus can have a genetic basis [Westendorp R G, Langermans J A, Huizinga T W, Verweij C L, Sturk A. Genetic influence on cytokine production in meningococcal disease. Lancet 1997; 349(9069):1912-3.]. Unfortunately, such heterogenic responses limit the utility of downstream acute-phase reactants, such as, for example, hsCRP as inflammatory marker for the risk stratification. In contrast to this, PlGF appears to be a direct proximal stimulus for inflammatory processes within the vascular wall [Luttun A, Tjwa M, Moons L, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumour angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 2002; 8(8):831-40.]. Indeed, elevated PlGF serum levels were extremely informative, specifically in patients with acute coronary syndromes, but not non-elevated hsCRP serum levels. In this cohort of patients, elevated PlGF serum levels identified a subgroup of patients with markedly elevated cardiac risk that was similar to the high-risk patients that were defined by elevated troponin serum levels. Thus, elevated PlGF serum levels can indeed represent primary inflammatory indicators of coronary instability.
Furthermore, since the pro-inflammatory effects of PlGF can be specifically inhibited by blocking its receptor Flt-1, these results can also provide an approach for a novel anti-inflammatory therapeutic target in patients with coronary arterial disease [Luttun A, Tjwa M, Canneliet P. Placental Growth Factor (PlGF) and Its Receptor Flt-1 (VEGFR-I): Novel Therapeutic Targets for Angiogenic Disorders. Ann N Y Acad Sci 2002; 979:80-93.]. The pro-inflammatory effects of PlGF can be specifically inhibited by blocking its receptor Flt-1, and provide a novel anti-inflammatory therapeutic option in patients with coronary arterial disease.
The results of the present study show that elevated blood levels of the metalloproteinase PAPP-A are associated with negative outcome in patients with ACS. In agreement with an examination that was performed only recently, the predictive value of PAPP-A plasma levels was most prominent in patients without an increase of troponin. Thus, an elevated PAPP-A plasma level is not only a marker of plaque-instability that promotes the development of ACS but, more important, indicates a bad prognosis, before the occurrence of an acute ischemic event that is caused by plaque-instability. In addition, elevated PAPP-A levels provide additional prognostic information, even in patients with elevated hsCRP plasma levels, suggesting that at least in some patients elevated hsCRP level are not associated with vascular inflammation. The result that the predictive values of PAPP-A were limited to patients with low levels of the anti-inflammatory cytokine interleukin-10 further supports the concept that the balance between pro- and anti-inflammatory cytokines is [important] for the outcome for the patients in ACS. Using a multivariate regression analysis, several biochemical markers, including troponin T, soluble CD40 ligand, interleukin-10, and PAPP-A were identified as independent predictive markers for the outcome for the patients during the subsequent six months of follow-up.
Cardiac troponines are sensitive and specific markers of myocardial necrosis, secondary to thrombotic complications during an acute coronary syndrome, and are highly predictive for the early clinical progression after the outbreak of ACS. However, the risk stratification in troponin negative patients with acute coronary syndrome remains challenging.
About two thirds of the patients with ACS, but without elevations of the ST-segment, exhibit normal values of Troponin, and more than half of the patients exhibit inconclusive electrocardiographical results. During the first weeks following the outbreak of an acute coronary syndrome the risk of mortality or non-fatal myocardial infarction in troponin-negative patients is at about 5 to 8%. Thus, the short term-occurrence of essential cardiovascular events remains relatively substantial in patients without evidences for myocardial necrosis, invoking the need for a further diagnostic processing. A continuous ST-segment monitoring, stress tests, and perfusion imaging methods can be of limited availability for the immediate risk stratification of patients from which it is assumed that they exhibit an acute coronary syndrome. The present study shows that PAPP-A levels indicate a subgroup of patients without elevation of troponin that during the early progression in time after the outbreak of symptoms exhibit an essentially higher risk for cardiac events (72 hours OR 3.17; 30 days: OR 3.33). In contrast to this, the patients without the modifications in the ST-segment that were both negative for TnT and PAPP-A, were subject to a very low risk (0.9% rate of occurrence). Thus, the determination of PAPP-A in patients with ACS is an effective tool for the short term-risk stratification of patients without elevated troponin level.
The progression and the subsequent destabilisation of arteriosclerotic plaques includes important changes in the structure of the arterial wall. The occurrence of a local state of inflammation in patients with ACS is well established as determined by inflammatory markers, such as, for example, CRP. Metalloproteinases are also potential indicators of arterial inflammation, and can contribute to the fragility of the lipid-rich arteriosclerotic plaques by degradation of extracellular matrix, and eventually to its rupture. As described earlier for several other metalloproteinases (MMP-1, MMP-3, MMP-12 or MMP-13) it was found for PAPP-A only very recently that it is expressed in eroded and released plaques, whereas the expression of PAPP-A could not be detected in stable plaques. Other studies have also shown that patients with hyperechonic or isoechoinic carotid plaques exhibit significantly elevated levels of PAPP-A than those with hypoechonic early carotid-lesions. The particular role of PAPP-A in the pathophysiology of ACS remains unclear. It was shown for PAPP-A that it is a specific activator of the insulin-like growth factor-I (IGF-I), a potent mediator of arteriosclerosis. As a matrix-metalloproteinase, PAPP-A could be involved in the processing of the extracellular matrix of plaques, and, consequently, affect the fibrous cap. This leads to a morphology of the plaque that is sensitive against erosion, rupture and subsequent thrombosis. The present study shows that that a single PAPP-A determination, obtained 8.7 hours after the occurrence of the symptoms provides a significant predictive value for the occurrence of death and non-fatal myocardial infarction during the following 6 months follow-up. These data suggest that PAPP-A plays an important pathophysiological role in the destabilisation of the arteriosclerotic plaques during ACS. The production of PAPP-A by activated cells within the arteriosclerotic lesions and its release into the extracellular matrix appears to be tightly associated with the local inflammatory process that occurs within the arterial wall as indicated by the significant positive correlation that was observed between CRP and PAPP-A levels. Indeed, the PAPP-A-levels were highly predictive in patients with elevated CRP levels, whereas in patients with low CRP levels, PAPP-A did not serve as a significant predictor for the outcome of the patients (FIG. 26a). Whilst CRP levels are connected with the troponin-increase, PAPP-A levels appear to be less sensitive against smaller myocardial injury, which could be of particular importance in patients with ACS, out of which at the time of arrival in the hospital about one third are positive for troponin. In addition, PAPP-A levels neither interfered with the predictive power of sCD40L, a marker of the activation of platelets in patients with ACS, nor did they affected these. By multivariate analysis PAPP-A, sCD40L and TnT all were found as independent predictors of adverse outcome (Table 2). A combination of PAPP-A and sCD40L was particularly obvious in patients that were negative for TnT, suggesting that both markers reflect distinct signal pathways reflect that eventually contribute to a pro-inflammatory and pro-coagulating milieu in the coronary circulation. Supporting for a complementary other than a competitive role in order to predict an adverse outcome in patients with ACS are the results of the inventors that the aggressive inhibition of the aggregation of the platelets by abciximab was particularly suitable in patients with elevated sCD40L levels.
In summary, the results of the present invention show that elevated plasma levels of PAPP-A as a marker of the vascular inflammation are associated with an elevated risk for subsequent cardiac events. The predictive value of PAPP-A plasma levels was independent from elevated troponin levels that reflect the actual risk secondary to thrombotic complications that lead to myocardial injury during an acute coronary syndrome. Thus, an elevated PAPP-A plasma level is not only a marker of the plaque instability with respect to the progression to a myocardial infarction, but also indicates a bad prognosis even after the occurrence of an acute ischemic event that is caused by plaque instability.
In summary, it can be stated that the present study documents the important and independent role of PlGF as a marker for the diagnostic and therapeutic risk stratification. The elevated cardiac risk of patients with high PlGF-concentrations that received a standard therapy with heparin and aspirin was reversed by the glycoprotein IIb/IIIa-receptor antagonist abciximab. The combined use of troponines and PlGF which both represent essential components of the pathophysiology in patients with acute coronary syndromes, provides important insights into the activity of the disease, the cardiologic risk, and the effectiveness of a treatment by means of glycoprotein IIb/IIIa-inhibition using abciximab that is superior to the use of a single marker.
In summary, the PlGF serum level represents an effective and reliable biomarker of the vascular inflammation, and negative outcome in patients with acute coronary syndromes. A determination of the PlGF serum levels significantly extends the predictive and prognostic information that is obtained from common inflammatory markers in acute coronary syndromes.